Abstract
Ion channel-based biosensors using label-free optical waveguide light-mode spectroscopy (OWLS) technique provide a sensitive measurement method of trans-channel ion transport, and allow further development in utilization of ion channels as models for pharmacological purposes (drug design targeting ion channels or diagnostic applications in clinical trials). This chapter describes a sensor setup for supported cell-derived membrane fragments deposited onto a hydrophilic polytetrafluoroethylene membrane with further separation from the OWLS sensor surface by a thin polyethylene terephthalate membrane. This approach provides spatial separation between the lipid layer and the sensor surface, and also allows space for possible extramembranous domains of the inbuilt membrane channel proteins. Influx of Cl− ions through GABAA channels in the presence or absence of GABA and channel blocking agent bicuculline is measured by changes of the optical characteristics in the evanescent field at near proximity of the OWLS sensor surface.
Key words
1 Introduction
The development of sensitive, real-time, and high-throughput approaches for screening of candidate substances affecting ion channels is becoming increasingly important in current rational drug design processes. Numerous drugs can act on membrane-embedded or membrane-associated proteins including membrane receptors, metabolite transporters, and ion channels. Optical biosensors are a highly effective tool in the characterization of such drug-protein interactions [1]. Incorporation of ion channels into the lipid bilayer on the sensor surface provides the platform for development of biosensors based on mimicking signals of ion channels in living cells. Ion channel-based biosensors are created either by self-assembly of pore-forming peptides of smaller molecular size such as gramicidin [2–4], melittin [5, 6], alamethicin [7, 8], and others [9, 10] or by incorporation of large transmembrane ion channel proteins [11, 12] onto the sensor surface. The biological activity of ion channels requires an appropriate lipid environment to preserve spatial conformation and proper conditions to allow assembling of the subunits of the multi-unit functional molecular complexes.
Artificial biomembrane constructions (liposome arrays, planar lipid bilayer, and supported lipid bilayer) have been developed and applied in multitude of techniques and experimental designs [13, 14]. The use of liposomes attached directly to the sensor surface in the biosensor approach presents some difficulties in the experiments (bulk composition effects), e.g., liposome motion, fusion or detachment, and ionic changes inside or outside of vesicles potentially generating significant changes in the refractive index and thus causing serious limitations to the rapid measurement of transport processes. An alternative approach to the use of liposomes for ion channel-ligand interaction studies is the application of planar lipid membranes. The artificial lipid layer platform enables variations of experimental conditions, but also challenges with several difficulties in sensor technology, including errors in the continuity of the lipid layers, as well as their mechanical instability and short lifetime. Lipid layer structures can be created by using the Langmuir-Blodgett method or by fusing liposomes on hydrophilic or hydrophobic solid surfaces [15]. The success of the preparation of supported lipid bilayers depends on several factors, such as surface charge, lipid composition, size of the liposomes, and other experimental conditions including pH and ionic strength [15, 16]. The significant drawback of the design based on solid supported lipid bilayers is that it cannot provide the appropriate space between the sensor surface and the lipid layer to accommodate extramembrane parts of transmembrane proteins. To overcome these shortcomings, several methodologies have been developed to place lipid layers at a distance from the sensor surface [17–19]. Due to their chemical stability, as well as variable porosity and hydrophobicity, Teflon polymers (polytetrafluoroethylene, PTFE) and copolymers (e.g., ethylene tetrafluoroethylene, ETFE) are well applicable as holders in supported lipid layer-based sensors. In situ-prepared Teflon films with multiple microfabricated pores were applied for the stable formation of planar lipid bilayers [7]. Alternatively, a porous Teflon surface was created on commercially available ETFE films using tungsten wire heated tips for pore formation [20]. Moreover, hydrophilic and hydrophobic Teflon filters with different pore sizes were also utilized as membrane holders to support the formation of artificial lipid bilayers with built-in functional ion channels [12, 21].
Ion flux through ion channels embedded in the lipid layer can be monitored by measuring electrochemical (as conductivity) or optical (as refractive index) parameters. The commonly used electrophysiological technique for studying ion channel activity and measuring the kinetics of ion channels is the patch clamp method pioneered by Neher and Sakman [22]. Optical detection-based techniques, such as surface plasmon resonance [23] or optical waveguide light-mode spectroscopy (OWLS) [24] sensors, detect physical changes of light in a narrow evanescent field over the sensor surface. Thus, these optical biosensors provide real-time information on molecular interactions without labeling of the interacting molecules [1].
Various techniques were utilized for lipid layer deposition [25, 26] and for monitoring functions of inbuilt ion channels [12, 27] in OWLS-based assay systems. This chapter describes an OWLS measurement system for detecting the channel functions of the GABAA (α5, β2, γ2) receptor in the presence or absence of γ-aminobutyric acid (GABA) and the competitive GABA-blocker bicuculline [12]. In the sensor design, the lipid bilayer is kept at a distance from the detection proximity of the sensor surface by inserting membrane sheets, so the lipid bilayer is outside of the sensing volume. The upper (PTFE) membrane towards the external medium supports the formation of lipid layers containing the ion channels to be investigated, and provides an environment for the extramembrane part of protein. The lipid layer, supported by the PTFE membrane, ideally provides full insulation by eliminating the bulk permeation of electrolytes. In real applications this insulation may be imperfect that may contribute to background signal intensity. The bottom (polyethylene terephthalate, PET) membrane facing towards the sensor excludes lipid vesicles from the detection field of the sensor, but allows passive migration of ions through the lipid layers favorably via the opening of ion channels to the sensor surface.
2 Materials
Reagents are available from Sigma-Aldrich (Hungary), unless stated otherwise. All buffer solutions are made in deionized distilled water (18.2 MΩ cm at 25 °C) and filtered through 0.22 μm Millex®GP filter (Millipore, Hungary) prior to use. Buffer solutions are stored at 4 °C and used within 1 month upon preparation.
2.1 Buffers
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1.
Artificial cerebrospinal fluid (ACSF): 145 mM NaCl, 3 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM d-glucose, 10 mM HEPES, pH 7.4.
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2.
Cl−-free ACSF: 140 mM Na-acetate, 5 mM KH2PO4, 0.8 mM MgSO4, 1.8 mM Ca-acetate, 10 mM d-glucose, 10 mM HEPES, pH 7.4 (see Note 1 ).
2.2 Reagents
2.2.1 Reagents for Liposome Preparation
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1.
Egg yolk lecithin (composition: 70 % phosphatidylcholine, 10 % phosphatidylethanolamine, and 20 % other lipids including neutral lipids; Avanti Polar Lipids, Alabaster, AL, USA).
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2.
Texas Red® DHPE (1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt; Invitrogen, Carlsbad, CA, USA).
2.2.2 Reagents for Cell Membrane Assay
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1.
Protease inhibitor cocktail tablets (Complete Mini, Roche, Hungary).
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2.
γ-Aminobutyric acid (GABA).
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3.
Bicuculline (see Note 2 ).
2.3 Membrane Sheets
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1.
PET membrane (RoTrac®; thickness 23 μm, with regular pores of 50 nm diameter; Oxyphen AG, Switzerland).
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2.
PTFE membrane (LCR; thickness 140 μm, virtual pore diameter 450 nm; Millipore, Hungary).
2.4 Cell Line
1. Cell lines expressing GABAA (α5, β2, γ2) receptors (see Note 3 ).
2.5 Equipment
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1.
OWLS 110 instrument with OW 2400 grating coupler sensors and BioSense 2.6 software (MicroVacuum Ltd, Budapest, Hungary).
3 Methods
3.1 Liposome Preparation
For cell membrane modeling it is very important to use liposomes consisting of single phospholipid bilayer—unilamellar vesicles. In this approach unilamellar liposomes are used for fusing with cell-derived membranes containing ion channels. The protocol below describes a simple and rapid method for liposome preparation from egg yolk lecithin according to Moscho et al. [28].
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1.
Dissolve egg yolk lecithin in chloroform-methanol (9:1) mixture at a concentration of 2 mg/ml.
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2.
Dissolve Texas Red DHPE in chloroform-methanol (9:1) mixture at a concentration of 1 mg/ml.
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3.
Add 1.1 μl of the abovementioned Texas Red DHPE solution to 2 ml of the egg yolk lecithin solution (see Note 4 ).
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4.
Add 2 ml of the lipid mixture to a 100 ml round-bottomed flask.
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5.
Layer 7 ml of the corresponding buffer above the organic solution (the density of the organic solution is higher than that of the buffer) and remove the organic solvent from the rotating flask immersed into a 30 °C water bath under reduced pressure (final vacuum < 20 mmHg) (see Note 5 ).
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6.
Dispense the remaining turbid suspension (~6 ml) into 1.5 ml Eppendorf tubes, and centrifuge at 2,085 × g for 10 min.
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7.
Collect the pellets and suspend them in 600 μl buffered saline (see Note 5 ).
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8.
Check the quality of the liposome preparation by phase-contrast or confocal fluorescence microscope. Avoid the high proportion of multilamellar liposomes. Liposomes prepared with this method contain mainly large (LUV) and giant (GUV) unilamellar vesicles.
3.2 Preparation of Cell Membrane Extracts
Instead of using ion channel proteins in purified form for insertion into liposomes or artificial lipid layers, cell-derived membrane fractions enriched genetically in a given transmembrane channel can be used. That helps multi-unit transmembrane channel to keep its native structure and activity. In the mammalian brain GABAA receptors are the major mediators of inhibitory neurotransmission. The GABA-gated ion channel upon activation selectively conducts Cl− through its pore. The GABAA receptor contains the binding site for GABA that also binds several drugs such as bicuculline. Moreover, the channel activity may be allosterically modulated by a number of drugs. The following protocol describes preparation of cellular membrane fractions from HEK293 cells expressing transmembrane GABAA (α5, β2, γ2) receptors.
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1.
Wash the adherent cells 3× with PBS.
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2.
Detach cells from the culture surface with 1 mM EDTA-PBS (pH 7.4).
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3.
Centrifuge 15 ml of the cell suspension (at least 6.7 × 106 cells/ml density) at 200 × g for 10 min at 4 °C (see Note 6 ).
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4.
Resuspend the pellets in tenfold volume (approximately 200 μl) of ice-cold buffered saline containing protease inhibitors (applied according to the manufacturer’s instruction).
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5.
Rupture cells by three freezing-thawing cycles using a dry ice for 2 min and 37 °C water bath for 5 min (see Note 7 ).
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6.
Centrifuge the suspension at 1,100 × g for 10 min at 4 °C to remove larger cell debris and nuclei.
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7.
Centrifuge supernatant at 21,000 × g for 20 min at 4 °C to sediment mitochondria (see Note 8 ).
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8.
Use the supernatant containing fragments of mixed cellular membranes in the OWLS assays (see Note 9 ).
3.3 Application of Cell-Derived Membrane Fraction onto the PTFE/PET Membranes
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1.
Mix 50 μl aliquot of the cell membrane fraction (obtained in Section 3.2) with equal volume of liposomes (obtained in Section 3.1) and incubate at room temperature for 2 h.
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2.
Cut out the PTFE and the PET membranes to appropriate size (12 mm × 8 mm) to fit the OWLS chip and put into the Cl−-free ACSF buffer.
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3.
Place a piece of PET membrane onto a sensor surface with carefully preventing ingress of air bubbles (see Note 10 ).
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4.
Layer a piece of the PTFE membrane above the PET membrane with carefully preventing ingress of air bubbles (see Note 10 ).
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5.
Place the OWLS chip with membranes into the sensor holder.
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6.
Inject 100 μl of mixed liposome-cell membrane suspension into the OWLS cuvette using Hamilton syringe, and incubate it for 2 h at room temperature (see Note 11 ).
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7.
After sedimentation, wash the cuvette with Cl−-free ACSF until stable NTM and NTE values.
3.4 Application of Compounds Affecting Ion Channel Activity
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1.
Prepare a solution of 100 μM GABA in ACSF buffer (Cl−-containing).
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2.
Prepare a solution of 100 μM bicuculline in ACSF buffer (Cl−-containing) (see Note 2 ).
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3.
Prepare a solution of 100 μM GABA and 100 μM bicuculline in ACSF buffer (Cl−-containing) (see Note 2 ).
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4.
Inject the above three test solutions consecutively into the flow stream of Cl−-free ACSF. Start each injection after the return of the original NTM and NTE baseline (Fig. 1).
3.5 OWLS Assay [29]
The OW 2400 sensor chip, used in the OWLS110 biosensor system, consists of a 12 mm × 8 mm substrate glass slide covered with a thin SiO2-TiO2 waveguide film (refractive index: nf = 1.77 ± 0.03) with a 12 mm × 2 mm optical grating (2,400 lines/mm). The optical grating incouples the light of a He-Ne laser at a given resonance angle into the waveguide layer [30, 31]. Total internal reflection of light creates an evanescent field in a small (typically 150–200 nm) sensing volume above the sensor surface, decreasing exponentially with the distance from the waveguide. Incoupling is a resonance phenomenon that occurs at two well-defined angles of incidence of the laser beam: one for transverse electric (TE) and the other for transverse magnetic (TM) mode. This angle depends on optical features of the sensor surface (optical grating on the surface and refractive index of the sensor layer) and on the refractive index of the medium covering the surface of the waveguide. By varying the angle of incidence of the laser light, the spectrum (both electric and magnetic modes) can be obtained, from which the effective refractive indices and, in turn, analyte concentrations in the medium are calculated [32]. To accelerate detection velocity, chose one side (positive or negative) of the obtained spectrum with two bigger peaks (NTM and NTE) and select a range of ±0.2° around the incoupling angles; thus 10 data points/min can be reached.
The glass sensor chip is placed on the sensor holder (type SH-0812-08) and is tightened to its sealing O-ring. The sensor holder forms a flow cell above the glass sensor with a volume of 12 μl. The glass sensor chip and the sensor holder form an integrated unit, which is placed in the OWLS instrument during measurement. All assays are carried out in a flow-injection system at continuous buffer flow at a rate of 23 μl/min, and 22 °C, with continuous recording of both NTE and NTM signals in the OWLS system in all individual experiments. Background signal corresponding to the refractive index of the Cl−-free ACSF buffer (baseline) is recorded in the absence of GABAA channel agonists or antagonists. Signal development upon injection of 100 μl aliquots of a given test solution into the running buffer stream through the injector valve is recorded in time, and signal intensity is followed in each injection experiment until the baseline is stabilized again (Fig. 1). From the measured mode spectra deposited mass, refractive indices, effective refractive indices, and thickness of deposited material on the sensor surface can be determined using BioSense application.
4 Notes
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1.
Warning: To avoid contamination of the Cl−-free ACSF buffer with Cl− ions, do not use HCl if acidification is required. Use NaHCO3 instead.
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2.
Prepare 100 mM stock solution of bicuculline in dimethyl sulfoxide (DMSO), and dissolve this stock solution 1:1,000 in ACSF buffer to obtain the working concentration of bicuculline of 100 μM. Thus, the maximal concentration of DMSO in the final working solution is 0.1 %, known as not affecting the measurement. Working solutions should be prepared and used on the same day due to the extreme unstability of bicuculline at physiological pH. Store stock solutions as aliquots in tightly sealed vials at −20 °C up to 1 month.
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3.
A GABAA-expressing cell line reported previously in this sensor format [12] was human embryonic kidney (HEK293) cell line, expressing α5, β2, and γ2 subunits of human GABAA receptors (established by researchers of EGIS Pharmaceutical Inc., Hungary). Human GABAA (α5, β2, γ2) receptor cell line can be also purchased from ChanTest (Cleveland, Ohio; Catalog #: CT6119).
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4.
Texas Red-labeled liposomes are used to check the lipid coverage of the holder membrane by fluorescence microscope. Store Texas Red desiccated at −20 °C, and protect Texas Red and Texas Red-labeled liposomes from light.
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5.
Prepare liposomes in the same buffer as the running buffer to avoid refractive index changes due to possible differences in ionic strength.
-
6.
The cell pellets can be frozen at this point if necessary or be directly used for membrane preparation.
-
7.
Alternatively, the cells may be homogenized.
-
8.
Purity of the preparation may be further improved by additional centrifugation of the supernatant at 100,000 × g for 1 h and subsequent resuspension of the pellet in buffer.
-
9.
If samples are not being analyzed immediately, store at −20 °C until assaying.
-
10.
Air bubbles in the system can dramatically change optical sensing. To avoid the ingress of air bubbles between the sensor surface and the PET membrane, as well as between the two (PET and PTFE) membranes, put one drop of Cl−-free ACSF buffer onto the sensor surface, and carefully layer the PET membrane on it; and again put another drop of the buffer onto the PET membrane, and carefully layer the PTFE membrane on it. To avoid desiccation of the membranes, assemble the cuvette immediately upon placing the membrane sheets.
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11.
The supported lipid bilayer prepared by this method, of course, does not eliminate the possibility of the presence of intact liposomes on the membrane support, despite the washing procedure that is supposed to remove all excess liposomes. This, however, does not represent a problem in the measurement process, as even unruptured liposomes improve the insulation of supported layer.
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Acknowledgments
The author expresses her sincere appreciation to her coworkers in the study that resulted in the original publication related to this protocol report. Particular thanks are due to Emilia Madarász (Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary), István Szendrő and Katalin Erdélyi (Microvacuum Ltd., Budapest, Hungary), Pál Gróf and Nóra Kaszás (Semmelweis University, Budapest, Hungary), as well as Ferenc A. Anthony, Balázs Mihalik, and Ágnes Pataki (EGIS Pharmaceutical Co., Budapest, Hungary) for their contribution, technical support, and helpful discussions in the OWLS technique, liposome preparation, and HEK293 cell line, expressing GABAA (α5, β2, γ2) receptors, respectively. The material support by Oxyphen GmbH (Zürich, Switzerland) by providing samples of RoTrack membranes is also acknowledged.
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Székács, I. (2015). Optical Waveguide Light-Mode Spectroscopy for Ion Channel Profiling. In: Fang, Y. (eds) Label-Free Biosensor Methods in Drug Discovery. Methods in Pharmacology and Toxicology. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2617-6_8
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